Cloning Of Laccase Gene From Irpex Lacteus And Correlated Sequence Analysis

Laccases is a kind of Cu-contained polyphenol oxidasc produced by many white-rot fungi. It can degrade lignin and phenoxy containing herbicide and wastewater in petroleum industry and affect toxic phenolic materials, so laccases can be widely applied in paper making industry, drinking processing and environment protection. Most of white-rot fungi propagate slowly in water and agglomerate quickly, so they can only live on the surface of water, these characteristic directly restrict their applied for wastewater treatment. Research on the cloning laccase structure gene of white-rot fungi significancant for cultivate engineering bacilli, which express high laccase activity of laccase in selected 34 strains were mensurated, The sequence of six white-rot fungi laccase gene have been cloned and analyzed. Main results as followed:(1) The activity of laccase from 34 selected white-rot fungi was mensurated. The results indicated that 14 strains have the activity of laccase, including Phellinus linteus, Pycnoporus cinnabarinus, Tyromyces zonatulus, Daedalea quercina, Lenzites tricdor, Climacocystis borealis, Lenzites betulina, Pycnoporus sanguineus, Fuscoporia olibqua, Irpex lacteal, Favolus alveolaris, Cerrena unicolor, 1999-32, Peniophora veluta.(2) The rapid methods of RNA and DNA extraction from mycelial fungi were established. Entire and high purity DNA and RNA can be isolated by these methods.(3) Utilizing the orthogonal design experiment method, the best response system of Irpes lacteus RT-PCR was confirmed: lOxBuffer 2.0 μL, MgCl₂2.0 mM, dNTPs 200 μM, Primer 2.0 μM, Template 12.0 ng/μL, Taq 1.25 U, Annealing 30 ℃.(4) The laccase structure gene of Irpes lacteus was cloned with 10 random-primers and 3 anchor-primers. The sequences were compared in GenBank database, looking for the homology genes. These results suggest that 5 genes of total 23 genes were the laccase genes or relevant isoenzyme genes. The cloning of laccase genetic fragment established the foundation for the acquisition of the total length gene.